Ceramides form the backbone of all sphingolipids, and they have emerged as important regulators and mediators of cellular stress responses. Recently, however, significant complexity has arisen in ceramide metabolism and biology. Therefore, the long-term goal of this proposal is to understand specific pathways of ceramide metabolism involved in cell stress responses. Recent work in our laboratory has focused on a newly identified family of enzymes; namely ceramide synthases (CerS), which demonstrate selective preferences for the fatty acyl chains that they incorporate into ceramide. In addition, very recent and exciting work in our laboratory using proteomic approaches has led us to discover CerS-interacting proteins, and one of the most credible and exciting of these proteins is the long chain fatty acyl CoA synthetase (ACSL5). In compelling preliminary data, we demonstrate that this interaction defines a novel pathway of ceramide metabolism by which ACSL5 provides the long chain fatty acids to acylate ceramides at the 1 position to generate O-acylceramides. We also demonstrate that this likely occurs by the diacylglycerol acyltransferase DGAT2. Evidence strongly suggests storage of O-acylceramides in lipid droplets (LD), and functionally, this causes sequestration of ceramide in a pool that is biologically inaccessible for regulation of cell death pathways. This proposal, therefore, addresses the hypothesis that Ceramide is metabolized to O-acylceramide by a previously unappreciated ACSL5/DGAT2 dependent mechanism and stored in LD and that this metabolic pathway is a key regulator of ceramide-mediated biologies. To test this hypothesis we propose the following specific aims: Aim 1. Define a novel pathway of ceramide metabolism to O- acylceramide by ACSL5/DGAT2 dependent mechanism. Aim 2. Define a role for O- acylceramides (in LD) in the regulation of ceramide-mediated responses. Aim 3. Determine the mechanism of CerS/ACSL5 protein-protein interaction. Taken together these studies will uncover a novel ceramide metabolic pathway to O-acylceramide by ACSL5 and DGAT2 with important implications for cell regulation.